CN112759632A

CN112759632A - Preparation method of Sylvestin

Info

Publication number: CN112759632A
Application number: CN202110136257.XA
Authority: CN
Inventors: 张伟; 赖仞; 段银; 吕秋敏; 文永均; 郭德文; 张治业
Original assignee: Kunming Longjin Pharmaceutical Co ltd; Kunming Institute of Zoology of CAS
Current assignee: Kunming Longjin Pharmaceutical Co ltd; Kunming Institute of Zoology of CAS
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2021-05-07
Anticipated expiration: 2041-02-01
Also published as: CN112759632B

Abstract

The invention belongs to the technical field of preparation methods of polypeptide medicines, and particularly relates to a preparation method of Sylvestin. The method adopts a solid-phase synthesis mode to synthesize Sylvestin by sections; the number of the segments is 2-4 segments, wherein the N-terminal segment contains at least 12 amino acid residues. The scheme provided by the invention obviously improves the purity of the crude product and the total yield of the product, and has wide practical value and application prospect.

Description

Preparation method of Sylvestin

Technical Field

The invention belongs to the technical field of preparation methods of polypeptide medicines, and particularly relates to a preparation method of Sylvestin.

Background

The forest leech antithrombotic peptide Sylvestin is a single-chain polypeptide encoded by a forest leech analgesic peptide gene, and has the molecular weight of 4790.5 daltons and the isoelectric point of 6.28. The peptide sequence of Sylvestin is: Thr-Ser-Glu-Pro-Val-Cys-Ala-Cys-Pro-Lys-Met-Leu-Phe-Trp-Val-Cys-Gly-Lys-Asp-Gly-Glu-Thr-Tyr-Thr-His-Pro-Cys-Ile-Ala-Lys-Cys-His-Asn-Val-Glu-His-Asp-Gly-Lys-Cys-Lys-OH.

Researches show that Sylvestin can inhibit FXIIa and kallikrein, and has extremely obvious antithrombotic and acute cerebral hemorrhage inhibiting effects; can be used for preparing FXIIa and kallikrein inhibitors and medicines for resisting thrombus and inhibiting acute cerebral hemorrhage.

At present, Sylvestin is prepared by means of genetic engineering generally, and the chemical synthesis has lower cost and simpler reaction condition. However, the chemical synthesis method of Sylvestin is not reported at present.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a preparation method of Sylvestin.

The preparation method of Sylvestin provided by the invention adopts a solid-phase synthesis mode to synthesize Sylvestin by sections; the number of the segments is 2-4 segments, wherein the N-terminal segment contains at least 12 amino acid residues.

In some embodiments, the number of segments is 4 segments, wherein:

the sequence of the fragment a is Thr-Ser-Glu-Pro-Val-Cys-Ala-Cys-Pro-Lys-Met-Leu-Phe,

the sequence of the fragment b is-Trp-Val-Cys-Gly-Lys-Asp-Gly-Glu,

the sequence of the fragment c is-Thr-Tyr-Thr-His-Pro-Cys-Ile-Ala-Lys-Cys,

the sequence of the fragment d is-His-Asn-Val-Glu-Val-Glu-His-Asp-Gly-Lys-Cys-Lys.

In other embodiments, the number of segments is 2 segments, wherein:

the sequence of the fragment a is Thr-Ser-Glu-Pro-Val-Cys-Ala-Cys-Pro-Lys-Met-Leu,

the sequence of the fragment b is-Phe-Trp-Val-Cys-Gly-Lys-Asp-Gly-Glu-Thr-Tyr-Thr-His-Pro-Cys-Ile-Ala-Lys-Cys-His-Asn-Val-Glu-Val-Glu-His-Asp-Gly-Lys-Cys-Lys.

In the embodiment of the invention, the Sylvestin is prepared by adopting a one-by-one access mode or a segmented access mode, and the result shows that the segmented access mode is more favorable for improving the yield and the purity of the synthesized product. In the scheme of segmented access, the segmentation mode and the selection of a protecting group influence the synthesis effect.

In the invention, the side chain protecting groups of Thr, Ser and Tyr are tBu; the side chain protecting groups of Glu and Asp are OtBu; side chain protecting groups of Cys, His and Asn are Trt; the side chain protecting groups of Lys and Trp are Boc.

In the present invention, the first amino acid Thr can be protected by tBu alone or by adding Boc protecting group to the N-terminus.

In some embodiments, therefore, the number of segments is 2,

the fragment a is X-Thr (tBu) -Ser (tBu) -Glu (OtBu) -Pro-Val-Cys (Trt) -Ala-Cys (Trt) -Pro-Lys (Boc) -Met-Leu-OH, wherein X is Fmoc or Boc;

the fragment b is Fmoc-Phe-Trp (Boc) -Val-Cys (Trt) -Gly-Lys (Boc) -Asp (OtBu) -Gly-Glu (OtBu) -Thr (tBu) -Tyr (tBu) -Thr (tBu) -His (Trt) -Pro-Cys (Trt) -Ile-Ala-Lys (Boc) -Cys (Trt) -His (Trt) -Asn (Trt) -Val-Glu (OtBu) -His (Trt) -Asp (OtBu) -Gly-Lys (Boc) -Cys (Trt) -Lys (Boc) -resin.

In other embodiments, the number of segments is 4,

the fragment a is Fmoc-Thr (tBu) -Ser (tBu) -Glu (OtBu) -Pro-Val-Cys (Trt) -Ala-

Cys(Trt)-Pro-Lys(Boc)-Met-Leu-Phe-OH；

The fragment b is Fmoc-Trp (Boc) -Val-Cys (Trt) -Gly-Lys (Boc) -Asp (OtBu) -Gly-Glu (OtBu) -OH;

the fragment c is Fmoc-Thr (tBu) -Tyr (tBu) -Thr (tBu) -His (Trt) -Pro-Cys (Trt) -Ile-Ala-Lys (Boc) -Cys (Trt) -OH;

the fragment d is Fmoc-His (Trt) -Asn (Trt) -Val-Glu (OtBu) -His (Trt) -Asp (OtBu) -Gly-Lys (Boc) -Cys (Trt) -Lys (Boc) -resin.

The research result of the invention shows that the quantity of the segments is not positively correlated with the yield. In all the fragmentation schemes Sylvestin was fragmented between amino acid 12 and amino acid 13, enabling the highest yields and purities to be obtained. The N-terminal protection with Boc is inferior to Fmoc.

The choice of resin also has an effect on the yield. In the scheme of the invention, the resin is hydroxyl resin. In embodiments of the invention, the resin is selected from Wang resin or HMP Linker resin. In some embodiments, the resin is an HMP Linker resin. In the coupling-by-coupling scheme, or the stepwise coupling scheme, the starting material for the C-terminus is Fmoc-Lys (Boc) -HMP Linker resin. In the embodiment of the invention, the amino substitution value of the resin is 0.2 mmol/g-0.8 mmol/g; in some embodiments, the resin has a substitution value of 0.3mmol/g to 0.5 mmol/g; in some embodiments, the resin has a substitution value of 0.3 mmol/g.

In the present invention, the segmented synthesis comprises: preparing fragments, coupling, acidolysis, cyclization, purification and desalting.

In the step of preparing the fragments, the fragments are prepared in a one-by-one coupling mode, and particularly, an Fmoc solid-phase polypeptide synthesis strategy is adopted.

In the coupling step:

the coupled coupling agents include HOBt and DIC; the molar ratio of HOBt to DIC in the coupling agent is (1-2): 1, in some embodiments, the molar ratio of HOBt to DIC is 1: 1.

In the coupling step, the molar ratio of the Fmoc-protected amino acid or the protected amino acid fragment to the resin is (1.2-6) to 1, preferably (2.5-3.5): 1; in some embodiments, the molar ratio of Fmoc-protected amino acid or protected amino acid fragment to resin is 3: 1.

The coupling step is carried out in a DMF solution, the coupling temperature is room temperature, and the coupling time is 120-300 min.

In the step of acid hydrolysis:

and (3) carrying out acidolysis on the Sylvestin resin, simultaneously removing the resin and side chain protecting groups, and carrying out oxidative cyclization to obtain a Sylvestin crude product.

In the invention, the acidolysis agent for acidolysis comprises trifluoroacetic acid, 1, 2-ethanedithiol and water; in the embodiment of the invention, the volume ratio of trifluoroacetic acid, 1, 2-ethanedithiol and water in the acidolysis agent is (80-95): (1-10): (1-10). In some embodiments, the volume ratio of trifluoroacetic acid, 1, 2-ethanedithiol and water in the acidolysis agent is (89-91): (4-6): (4-6). In some embodiments, the volume ratio of trifluoroacetic acid, 1, 2-ethanedithiol, and water in the acid hydrolysis agent is 95:5: 5.

The acidolysis step comprises: mixing Sylvestin resin with an acidifier for reaction to obtain the linear-chain Sylvestin peptide. Preferably, the product after reaction is washed with TFA.

In the acidolysis step, the mass-volume ratio of Sylvestin resin to acidolysis agent is 1 g: 4ml to 15 ml; in some embodiments, the mass-to-volume ratio of Sylvestin resin to acid disintegrant is 1 g: 9ml to 11 ml; in some embodiments, the mass-to-volume ratio of Sylvestin resin to acid disintegrant is 1 g: 10 ml.

The acidolysis conditions of the invention are as follows: and (3) carrying out acidolysis for 1-5 hours at room temperature, preferably for 2 hours at room temperature.

In the cyclization step of the present invention:

in the cyclization step, iodine and H are used₂O₂Or at least one of DMSO is an oxidant, and sulfydryl of 6 cysteines in the Sylvestin peptide sequence is oxidized and paired to form stable tricyclic peptide. The cyclized oxidant is iodine and H₂O₂Or DMSO, preferably DMSO. In the present example, DMSO was used as the oxidizing agent. The step of cyclizing comprises: and (3) resuspending the linear Sylvestin peptide into a concentration of 1.5mg/ml by using a buffer solution, adjusting the pH value to 7.5, and performing oxidation reaction overnight to obtain a cyclized crude Sylvestin product. The buffer consisted of water, 1mmol/L cysteine hydrochloride and 15 vol% DMSO.

In the purification step of the present invention:

the purification adopts high performance liquid chromatography; the chromatographic column is a reversed phase C18 chromatographic column, the diameter of the filler is 10 μm, and the size of the chromatographic column is 77mm × 250 mm.

The mobile phase a is 100mmol/LNH₄AC.H₂O PH＝7.0±0.2，

Mobile phase b was acetonitrile.

The flow rate of the mobile phase is 90mL/min, and the elution procedure of the purification chromatography is as follows:

serial number	Time (min)	B％
			1	0.00	8％
2	10.00	8％
			3	25.00	14±2％
4	40.00	14±2％
			5	45.00	17±2％
6	55.00	17±2％
			7	60.00	27±2％
8	70.00	27±2％
			9	70.50	80％
10	73.50	80％
			11	74.00	5％
12	86.00	5％

In the desalting step of the present invention:

the desalting is carried out by high performance liquid chromatography, wherein the chromatographic column is a reversed phase C18 chromatographic column, the diameter of the filler is 10 μm, and the size of the chromatographic column is 77mm × 250 mm.

The mobile phase A is 1 wt% acetic acid water solution, and the mobile phase B is acetonitrile.

The flow rate of the mobile phase is 90mL/min, and the elution procedure of the desalting chromatography is as follows:

after the desalting step, the obtained eluent is subjected to reduced pressure concentration and freeze drying to obtain Sylvestin pure powder.

The preparation method of Sylvestin provided by the invention adopts a solid-phase synthesis mode to synthesize Sylvestin by sections; the number of the segments is 2-4 segments, wherein the N-terminal segment contains at least 12 amino acid residues. The scheme provided by the invention obviously improves the purity of the crude product and the total yield of the product, and has wide practical value and application prospect.

Detailed Description

The invention provides a preparation method of Sylvestin, and a person skilled in the art can use the content for reference and appropriately improve the process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The N-terminal fragment of the present invention refers to the fragment closest to the N-terminus among several fragments synthesized by fragmentation. For example, the peptide chain of Sylvestin is divided into 2 segments, which are an N-terminal segment and a C-terminal segment. The peptide chain of Sylvestin is divided into 3 segments, namely an N-terminal segment, a middle segment and a C-terminal segment. The peptide chain of Sylvestin is divided into 4 segments, namely an N-terminal segment, a middle segment 1, a middle segment 2 and a C-terminal segment.

The "protecting group" or "protecting group" as referred to herein has its ordinary meaning in the art. A protecting group includes a chemical moiety that is attached to or configured to attach to a reactive group (i.e., a protected group) within a molecule (e.g., a peptide) such that the protecting group prevents or otherwise inhibits the protected group from participating in a reaction. Protection may be performed by attaching a protecting group to the molecule. Deprotection can occur when a protecting group is removed from a molecule, for example, by chemical transformation to remove the protecting group.

In the present invention, the "polypeptide" or "peptide" has its ordinary meaning in the art and may refer to an amide from two or more aminocarboxylic acid molecules (the same or different) that forms a covalent bond by formally losing water from the carbonyl carbon of one aminocarboxylic acid molecule and the nitrogen atom of another aminocarboxylic acid molecule. "amino acid residue" also has its ordinary meaning in the art and refers to the composition of an amino acid (as a single amino acid or as part of a peptide) after it is combined with a peptide, another amino acid, or an amino acid residue. Generally, when an amino acid is combined with another amino acid or amino acid residue, water is removed, and the remaining amino acid is referred to as an amino acid residue. The term "amino acid" also has its ordinary meaning in the art and can include both proteinogenic amino acids and non-proteinogenic amino acids. The abbreviations for amino acid residues in the present invention are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

The term "coupling" or "grafting" as used herein refers to the process of adding a new amino acid or peptide fragment to a bound amino acid or peptide. The test materials adopted by the invention are all common commercial products and can be purchased in the market. In the specific embodiment of the present invention, the English abbreviations used in the application documents have the corresponding Chinese meanings as shown in the following table.

English abbreviation	Name of Chinese	English abbreviation	Name of Chinese
				Fmoc	9-fluorenylmethoxycarbonyl group	OtBu	Tert-butoxy radical
tBu	Tert-butyl radical	Boc	Boc-acyl
				Trt	Trityl radical	Leu	Leucine
Ser	Serine	Phe	Phenylalanine
				Glu	Glutamic acid	Thr	Threonine
Trp	Tryptophan	Arg	Arginine
				Asp	Aspartic acid	Gln	Glutamine
Ala	Alanine	Ile	Isoleucine
				Tyr	Tyrosine	His	Histidine
Gly	Glycine	Lys	Lysine
				Val	Valine	Pro	Proline

The invention is further illustrated by the following examples:

EXAMPLE 1 Synthesis of Sylvestin peptide resin by fragment grafting

Preparation of fragments

Fragment a (amino acids 1-12):

Boc-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Pro-Val-Cys(Trt)-Ala-Cys(Trt)-Pro-Lys(Boc)-Met-Leu-OH

the preparation method adopts Fmoc solid-phase polypeptide synthesis strategy, and comprises the following steps:

dissolving 0.09mol of Fmoc-Met-OH and 0.09mol of HOBt with a proper amount of DMF; and adding 0.09mol of DIC slowly into the DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain activated Fmoc-Met-OH for later use.

0.03mol of Fmoc-Leu-2-Cl-Trt resin is taken, deprotected by 20 percent PIP/DMF solution for 25 minutes, washed and filtered to obtain the Fmoc-removed Leu-2-Cl-Trt resin.

And adding the activated Fmoc-Met-OH solution into the Fmoc-removed Leu-2-Cl-Trt resin (the molar ratio is 3:1), performing coupling reaction for 120-300 minutes, filtering and washing to obtain the Fmoc-Met-Leu-2-Cl-Trt resin.

The fragment A resin was prepared by coupling Fmoc-Lys (Boc) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Ala-OH, Fmoc-Cys (Trt) -OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH and Boc-Thr (tBu) -OH in this order according to the above reaction conditions.

Fragment a resin with 1% TFA/DCM solution (10ml/g resin) at room temperature cleavage, collecting lysate, vacuum concentration of the obtained precipitate with water washing 5 times, at 35 degrees C under reduced pressure drying fragment a. Purity 80.9%, mass spectrum should be 1156.4.

Fragment b (amino acids 13-43):

Fmoc-Phe-Trp (Boc) -Val-Cys (Trt) -Gly-Lys (Boc) -Asp (OtBu) -Gly-Glu (OtBu) -Thr (tBu) -Tyr (tBu) -Thr (tBu) -His (Trt) -Pro-Cys (Trt) -Ile-Ala-Lys (Boc) -Cys (Trt) -His (Trt) -Asn (Trt) -Val-Glu (OtBu) -His (Trt) -Asp (OtBu) -Gly-Lys (Boc) -Cys (Trt) -Lys (Boc) -HMP Linker resin

dissolving 0.03mol of Fmoc-Cys (Trt) -OH and 0.03mol of HOBt in a proper amount of DMF; and adding 0.03mol of DIC slowly into the DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain activated Fmoc-Cys (Trt) -OH for later use.

0.01mol of Fmoc-Lys (Boc) -HMP Linker resin (substitution value about 0.3mmol/g) was deprotected with 20% PIP/DMF solution for 25 min, washed and filtered to give Fmoc-removed resin.

Adding the activated Fmoc-Cys (Trt) -OH solution into the Fmoc-removed resin (the molar ratio is 3:1), performing coupling reaction for 120-300 minutes, and filtering and washing to obtain the resin containing Fmoc-Cys (Trt) -Lys (Boc) -HMP Linker.

Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-His (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Cys (Trt) -OH, Fmoc-Pro-OH, Fmoc-His (Trt) -OH, Fmoc-ThrtBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-ThrtBu (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Cys (Trt) -OH, Fmoc-Val-OH, Fmoc-Trp (Boc) -OH and Fmoc-Phe-OH are used as raw materials for coupling to prepare a fragment b resin.

Two, fragment coupling

Dissolving 0.03mol of the fragment a and 0.03mol of HOBt by using a proper amount of DMF; and adding 0.03mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain activated fragment a for later use.

0.01mol of fragment b (substitution value about 0.3mmol/g) was taken, deprotected with 20% PIP/DMF solution for 25 min, washed and filtered to give Fmoc-removed fragment b resin.

Adding the activated fragment a solution into a fragment b resin (the molar ratio of the fragment a to the fragment b is 3:1), performing coupling reaction for 120-300 minutes, filtering and washing to obtain Sylvestin peptide resin: Boc-Thr (tBu) -Ser (tBu) -Glu (OtBu) -Pro-Val-Cys (Trt) -Ala-Cys (Trt) -Pro-Lys (Boc) -Met-Leu-Phe-Trp (Boc) -Val-Cys (Trt) -Gly-Lys (Boc) -Asp (OtBu) -Gly-Glu (OtBu) -Thr (tBu) -Tyr (tBu) -Thr (tBu) -His (Trt) -Pro-Cys (Trt) -Ile-Ala-Lys (Boc) -Cys (Trt) -His (Trt) -Asn (Trt) -Val-Glu (OtBu) -His (Trt) -Asp (OtBu) -Gly-Boc) -Lys (Trt) -Lys (HMP LinTak).

Example 2 Synthesis of Sylvestin peptide resin by fragment grafting

Preparation of fragments

Fragment a (amino acids 1-12):

Fmoc-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Pro-Val-Cys(Trt)-Ala-Cys(Trt)-Pro-Lys(Boc)-Met-Leu-OH

the preparation method is the same as example 1, the purity is 85.0%, and the mass spectrum is 1278.6.

Fragment b was prepared as in example 1,

preparation of resin for obtaining Fmoc-Phe-Trp (Boc) -Val-Cys (Trt) -Gly-Lys (Boc) -Asp (OtBu) -Gly-Glu (OtBu) -Thr (tBu) -Tyr (tBu) -Thr (tBu) -His (Trt) -Pro-Cys (Trt) -Ile-Ala-Lys (Boc) -Cys (Trt) -His (Trt) -Asn (Trt) -Val-Glu (OtBu) -His (Trt) -Asp-OtBu) -Gly-Lys (Boc) -Cys Trt- (Lys (Boc) -HMP Linker

Two, fragment coupling

And adding the activated fragment a solution into a fragment b resin (the molar ratio of the fragment a to the fragment b is 3:1), performing coupling reaction for 120-300 minutes, and filtering and washing to obtain the Fmoc-Sylvestin peptide resin.

Finally, deprotection was carried out for 25 minutes using 20% PIP/DMF solution, washed and filtered to obtain Sylvestin peptide resin.

Example 3 Synthesis of Sylvestin peptide resin by fragment grafting

Preparation of fragments

Fragment a (amino acids 1-13): Fmoc-Thr (tBu) -Ser (tBu) -Glu (OtBu) -Pro-Val-

Cys(Trt)-Ala-Cys(Trt)-Pro-Lys(Boc)-Met-Leu-Phe-OH

Fragment b (amino acids 14-21): Fmoc-Trp (Boc) -Val-Cys (Trt) -Gly-Lys (Boc) -Asp (OtBu) -Gly-Glu (OtBu) -OH

Fragment c (amino acids 22-31): Fmoc-Thr (tBu) -Tyr (tBu) -Thr (tBu) -His (Trt) -Pro-Cys (Trt) -Ile-Ala-Lys (Boc) -Cys (Trt) -OH

Fragment d (amino acids 32-43): Fmoc-His (Trt) -Asn (Trt) -Val-Glu (OtBu) -His (Trt) -Asp (OtBu) -Gly-Lys (Boc) -Cys (Trt) -Lys (Boc) -HMP Linker resin

Wherein the fragments a, B and c are prepared by the same method as the fragment A in example 1, and the fragment d is prepared by the same method as the fragment B in example 1.

Two, fragment coupling

Dissolving 0.03mol of the fragment c and 0.03mol of HOBt in a proper amount of DMF; and adding 0.03mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain activated fragment c for later use.

0.01mol of fragment d (substitution value about 0.3mmol/g) was taken, deprotected with 20% PIP/DMF solution for 25 minutes, washed and filtered to give Fmoc-removed fragment d resin.

And adding the activated fragment c solution into a fragment d resin (the molar ratio of the fragment A to the fragment B is 3:1), performing coupling reaction for 120-300 minutes, filtering and washing, and then coupling the fragment B and the fragment a according to the same method to obtain the Fmoc-Sylvestin peptide resin.

Example 4 Synthesis of Sylvestin peptide resin by one-by-one Access method

Coupling with Fmoc-Cys (Trt) -OH using Fmoc-Lys (Boc) -HMP Linker resin as starting resin, the method comprising:

Adding the activated Fmoc-Cys (Trt) -OH solution into the Fmoc-removed resin, performing coupling reaction for 120-300 minutes, and filtering and washing to obtain the resin containing Fmoc-Cys (Trt) -Lys (Boc) -HMP Linker.

Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-His (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Cys (Trt) -OH, Fmoc-Pro-OH, Fmoc-His (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Tyr tBu) -OH, (Fmoc-Thr) -OH, Fmoc-OtBu) -OH, Fmoc-Thr-OH, Fmoc-Glu-Tyr (Tyr-Thr) -OH, Fmoc, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Cys (Trt) -OH, Fmoc-Val-OH, Fmoc-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Ala-OH.H2O, Fmoc-Cys (Trt) -OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Boc-Thr (tBu) -OH are used as raw materials for coupling to prepare the Sylvin peptide resin.

EXAMPLE 5 preparation of crude Sylvestin

The Sylvestin peptide resin prepared in the example 1 is taken, a cracking reagent (10mL/g resin of the cracking reagent) with the volume ratio of TFA, water and EDT being 95:5 is added, the mixture is stirred evenly and reacted for 3 hours at room temperature, the reaction mixture is filtered by a sand core funnel, the filtrate is collected, the resin is washed for 3 times by a small amount of TFA, the filtrate is combined and concentrated under reduced pressure, absolute ethyl ether is added for precipitation, the absolute ethyl ether is used for washing the precipitation for 3 times, the precipitation is dried for 6 hours under reduced pressure at 25 to 35 ℃, the precipitation is dissolved and diluted to a solution of 1.5mg/mL by using 1mmol/L cysteine hydrochloride 15% DMSO buffer solution, the pH value is adjusted to 7.5 by using ammonia water, the reaction is stirred overnight,

obtaining a crude Sylvestin solution, wherein the purity of the crude product is 58.5%.

EXAMPLE 6 preparation of crude Sylvestin

Taking Sylvestin peptide resin prepared in example 2, adding a cracking reagent (10mL/g resin cracking reagent) with the volume ratio of TFA, water and EDT being 95:5, stirring uniformly, stirring at room temperature for reaction for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, drying at 25-35 ℃ under reduced pressure for 6 hours, dissolving the precipitate with 1mmol/L cysteine hydrochloride 15% DMSO buffer solution, diluting to a solution of 1.5mg/mL, adjusting pH to 7.5 by using ammonia water, and stirring for reaction overnight to obtain a Sylvestin crude product solution with the purity of 63.1%.

EXAMPLE 7 preparation of crude Sylvestin

Taking Sylvestin peptide resin prepared in example 3, adding a cracking reagent (10mL/g resin cracking reagent) with the volume ratio of TFA, water and EDT being 95:5, stirring uniformly, stirring at room temperature for reaction for 3 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin by using a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate by using anhydrous ether for 3 times, drying at 25-35 ℃ under reduced pressure for 6 hours, dissolving the precipitate by using 1mmol/L cysteine hydrochloride 15% DMSO buffer solution, diluting to be a solution of 1.5mg/mL, adjusting pH to 7.5 by using ammonia water, stirring for reaction overnight, and obtaining a crude Sylvestin solution with the crude purity of 39.1%.

EXAMPLE 8 preparation of crude Sylvestin

Taking Sylvestin peptide resin prepared in example 4, adding a cracking reagent (10mL/g resin cracking reagent) with the volume ratio of TFA, water and EDT being 95:5, stirring uniformly, stirring at room temperature for reaction for 3 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin by using a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate by using anhydrous ether for 3 times, drying at 25-35 ℃ under reduced pressure for 6 hours, dissolving the precipitate by using 1mmol/L cysteine hydrochloride 15% DMSO buffer solution, diluting to be a solution of 1.5mg/mL, adjusting pH to 7.5 by using ammonia water, stirring for reaction overnight, and obtaining a crude Sylvestin solution with the crude purity of 27.9%.

EXAMPLE 9 purification of crude Sylvestin

Filtering the crude Sylvestin solution prepared in the embodiment 5 by using a 0.45-micron mixed microporous filter membrane, and purifying for later use;

purification was carried out by high performance liquid chromatography using a 10 μm reverse phase C18 as the chromatographic packing and 100mmol ammonium acetate/water solution-acetonitrile as the mobile phase. The flow rate of a chromatographic column of 77mm multiplied by 250mm is 90mL/min, a gradient system is adopted for elution, the sample is circularly injected and purified, a crude product solution is taken to be loaded in the chromatographic column, the mobile phase elution is started, a main peak is collected, acetonitrile is evaporated, and then a 0.45 mu m filter membrane is used for filtration, so that Sylvestin purified intermediate concentrated solution is obtained for desalination; gradient conditions for purification were:

desalting by high performance liquid chromatography with 1% acetic acid water solution-acetonitrile as mobile phase system, reverse phase C18 with purification chromatographic filler of 10 μm, and chromatographic column flow rate of 77mm × 250mm of 90 mL/min; the method comprises the steps of adopting a gradient elution and circulation loading method, loading a sample into a chromatographic column, starting mobile phase elution, collecting a map, observing the change of absorbance, collecting a main desalting peak, detecting the purity by using an analytical liquid phase, combining solutions of the main desalting peaks, concentrating under reduced pressure to obtain a Sylvestin aqueous solution, and freeze-drying to obtain a Sylvestin pure product 10.5g, wherein the purity is 98.9%, the maximum single impurity content is 0.12%, the total yield is 21.9%, and the molecular weight is 4789.5 (100% M + H). Gradient conditions for purification were:

serial number	Time (min)	B％
			1	0.00	5％
2	35.00	5％
			3	36.00	40±2％
4	60.00	40±2％
			5	60.50	80％
6	64.50	80％
			7	65.00	5％
8	77.00	5％

EXAMPLE 10 purification of crude Sylvestin

Filtering the crude Sylvestin solution prepared in the embodiment 6 by using a 0.45-micron mixed microporous filter membrane, and purifying for later use;

purification was performed by high performance liquid chromatography using 10 μm reverse phase C18 as the chromatographic packing, alternating between two mobile phase systems, the first being 0.1% TFA/water-0.1% TFA acetonitrile and the second being 50mmol ammonium acetate/water-acetonitrile. The flow rate of a chromatographic column of 77mm multiplied by 250mm is 90mL/min, a gradient system is the same as that in the embodiment 9, the sample is circularly injected and purified, a crude product solution is taken to be loaded in the chromatographic column, the mobile phase elution is started, a main peak is collected, acetonitrile is evaporated, and then the solution is filtered by a filter membrane of 0.45 mu m, so that a Sylvestin purified intermediate concentrated solution is obtained for desalination;

desalting by high performance liquid chromatography with 1% acetic acid water solution-acetonitrile as mobile phase system, reverse phase C18 with purification chromatographic filler of 10 μm, and chromatographic column flow rate of 77mm × 250mm of 90 mL/min; the gradient system was the same as in example 9, the sample was loaded onto a chromatographic column, mobile phase elution was started, spectra were collected, changes in absorbance were observed, the desalted main peak was collected and the purity was checked by analytical liquid phase, the desalted main peak solutions were combined, concentrated under reduced pressure to give a Sylvestin aqueous solution, which was freeze-dried to give 11.6g of a pure Sylvestin product with a purity of 98.5%, 0.15% of the maximum single impurity, a total yield of 24.2%, and a molecular weight of 4789.5 (100% M + H).

EXAMPLE 11 purification of crude Sylvestin

Filtering the crude Sylvestin solution prepared in the embodiment 7 by using a 0.45-micron mixed microporous filter membrane, and purifying for later use;

purification was performed by high performance liquid chromatography using reverse phase C18 with 10 μm chromatography packing and alternating purification with two mobile phase systems, the first being 0.1% TFA/water-0.1% TFA/acetonitrile and the second being 50mmol ammonium acetate/water-acetonitrile. The flow rate of a chromatographic column of 77mm multiplied by 250mm is 90mL/min, a gradient system is the same as that in the embodiment 9, the sample is circularly injected and purified, a crude product solution is taken to be loaded in the chromatographic column, the mobile phase elution is started, a main peak is collected, acetonitrile is evaporated, and then the solution is filtered by a filter membrane of 0.45 mu m, so that a Sylvestin purified intermediate concentrated solution is obtained for desalination;

desalting by high performance liquid chromatography with 1% acetic acid water solution-acetonitrile as mobile phase system, reverse phase C18 with purification chromatographic filler of 10 μm, and chromatographic column flow rate of 77mm × 250mm of 90 mL/min; the gradient system was performed as in example 9, the sample was loaded onto a chromatographic column by a cyclic loading method, the mobile phase elution was started, the spectrum was collected, the change in absorbance was observed, the desalted main peak was collected and the purity was measured by an analytical liquid phase, the desalted main peak solutions were combined, concentrated under reduced pressure to give a Sylvestin aqueous solution, which was freeze-dried to give 3.2g of a pure Sylvestin product, having a purity of 98.2%, a maximum single impurity of 0.49%, a total yield of 6.7%, and a molecular weight of 4789.6 (100% M + H).

EXAMPLE 12 purification of crude Sylvestin

Taking the crude Sylvestin solution prepared in the embodiment 8, filtering the solution by using a 0.45-micron mixed microporous filter membrane, and purifying for later use;

purification was performed by high performance liquid chromatography using reverse phase C18 with 10 μm chromatography packing and alternating purification with two mobile phase systems, the first being 0.1% TFA/water-0.1% TFA/acetonitrile and the second being 50mmol ammonium acetate/water-acetonitrile. The flow rate of a chromatographic column of 77mm multiplied by 250mm is 90mL/min, a gradient system is adopted, the same example as the example 9 is adopted, the sample is circularly injected and purified, a crude product solution is taken to be loaded in the chromatographic column, the mobile phase elution is started, a main peak is collected, acetonitrile is evaporated, and then the solution is filtered by a 0.45 mu m filter membrane to obtain a Sylvestin purified intermediate concentrated solution for desalination;

desalting by high performance liquid chromatography with 1% acetic acid water solution-acetonitrile as mobile phase system, reverse phase C18 with purification chromatographic filler of 10 μm, and chromatographic column flow rate of 77mm × 250mm of 90 mL/min; the gradient system is the same as the embodiment 9, the sample loading method is circulated, the sample is loaded in a chromatographic column, the mobile phase elution is started, the atlas is collected, the change of the absorbance is observed, the desalted main peak is collected and the purity is detected by the analysis liquid phase, the desalted main peak solution is combined, the pressure reduction concentration is carried out, the Sylvestrin water solution is obtained, the freeze drying is carried out, the Sylvestrin pure product 1.7g is obtained, the purity is 97.3 percent, the maximum single impurity is 1.53 percent, the total yield is 3.5 percent, and the molecular weight is 4789.5(100 percent M)⁺H)。

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

The preparation method of Sylvestin adopts a solid-phase synthesis mode, and prepares Sylvestin by segmented access;

the number of the segments is 2-4 segments, wherein the N-terminal segment contains at least 12 amino acid residues.
2. The production method according to claim 1,

the number of segments is 2 segments, wherein:

the sequence of the fragment a is Thr-Ser-Glu-Pro-Val-Cys-Ala-Cys-Pro-Lys-Met-Leu,

the sequence of the fragment b is-Phe-Trp-Val-Cys-Gly-Lys-Asp-Gly-Glu-Thr-Tyr-Thr-His-Pro-Cys-Ile-Ala-Lys-Cys-His-Asn-Val-Glu-Val-Glu-His-Asp-Gly-Lys-Cys-Lys;

the number of segments is 4 segments, wherein:

the sequence of the fragment a is Thr-Ser-Glu-Pro-Val-Cys-Ala-Cys-Pro-Lys-Met-Leu-Phe,

the sequence of the fragment b is-Trp-Val-Cys-Gly-Lys-Asp-Gly-Glu,

the sequence of the fragment c is-Thr-Tyr-Thr-His-Pro-Cys-Ile-Ala-Lys-Cys,

the sequence of the fragment d is-His-Asn-Val-Glu-Val-Glu-His-Asp-Gly-Lys-Cys-Lys.
3. The production method according to claim 2,

the fragment a is X-Thr (tBu) -Ser (tBu) -Glu (OtBu) -Pro-Val-Cys (Trt) -Ala-Cys (Trt) -Pro-Lys (Boc) -Met-Leu-OH, wherein X is Fmoc or Boc;

the fragment b is Fmoc-Phe-Trp (Boc) -Val-Cys (Trt) -Gly-Lys (Boc) -Asp (OtBu) -Gly-Glu (OtBu) -Thr (tBu) -Tyr (tBu) -Thr (tBu) -His (Trt) -Pro-Cys (Trt) -Ile-Ala-Lys (Boc) -Cys (Trt) -His (Trt) -Asn (Trt) -Val-Glu (OtBu) -His (Trt) -Asp (OtBu) -Gly-Lys (Boc) -Cys (Trt) -Lys (Boc) -resin.
4. The production method according to claim 2,

the fragment a is Fmoc-Thr (tBu) -Ser (tBu) -Glu (OtBu) -Pro-Val-Cys (Trt) -Ala-

Cys(Trt)-Pro-Lys(Boc)-Met-Leu-Phe-OH；

The fragment b is Fmoc-Trp (Boc) -Val-Cys (Trt) -Gly-Lys (Boc) -Asp (OtBu) -Gly-Glu (OtBu) -OH;

the fragment c is Fmoc-Thr (tBu) -Tyr (tBu) -Thr (tBu) -His (Trt) -Pro-Cys (Trt) -Ile-Ala-Lys (Boc) -Cys (Trt) -OH;

the fragment d is Fmoc-His (Trt) -Asn (Trt) -Val-Glu (OtBu) -His (Trt) -Asp (OtBu) -Gly-Lys (Boc) -Cys (Trt) -Lys (Boc) -resin.
5. The method of claim 3 or 4, wherein the resin is a hydroxyl resin, including Wang resin or HMP Linker resin.
6. The method according to claim 5, wherein the resin has an amino substitution value of 0.2 to 0.8 mmol/g.
7. The preparation method according to any one of claims 1 to 6, wherein the segmented access preparation of Sylvestin comprises: preparing fragments, coupling, acidolysis, cyclization, purification and desalting;

the coupled coupling agents include HOBt and DIC;

the acidolysis agent for acidolysis comprises trifluoroacetic acid, 1, 2-ethanedithiol and water;

the cyclized oxidant is iodine and H₂O₂Or DMSO, preferably DMSO.
8. The production method according to claim 6,

the molar ratio of HOBt to DIC in the coupling agent is (1-2): 1;

the volume ratio of trifluoroacetic acid, 1, 2-ethanedithiol and water in the acidolysis agent is (80-95): (1-10): (1-10).